Obesity has reached epidemic proportions in the U.S. and plays a major role in the development of type 2 diabetes, dyslipidemia, and cardiovascular disease. While most weight loss agents rely on suppressing appetite to reduce caloric intake, strategies that can safely enhance metabolic rate, a previously unrealized approach to weight loss or weight maintenance, have potential to effectively treat obesity. Brown adipocytes have an extremely high metabolic rate because they express mitochondrial uncoupling protein-1 (UCP1). This protein dissipates the electrochemical gradient in the mitochondria of brown adipocytes as heat. Brown adipose tissue (BAT) thermogenesis is increased upon exposure to low temperatures, and plays an important role in the maintenance of body temperature and energy balance in rodents. BAT is also a flexible tissue that normally enlarges or atrophies over time depending on environmental temperature. In many different rodent models, enhancement of BAT mass has convincingly been shown to lead to weight loss and diabetes resistance. While BAT was until recently thought to be effectively nonexistent in adult humans, recent data obtained with PET imaging shows that adults in fact have significant BAT, and that this tissue is functional. Other data demonstrates that the amount of active BAT in individuals is strongly correlated with leanness. Until recently no brown adipocyte stem cell had been identified. We discovered a population of human skeletal muscle-resident brown adipocyte progenitors that under appropriate conditions become fully functional brown adipocytes. Following differentiation, these cells have high levels of UCP1 and a very high metabolic rate. Using the cells we developed a high-throughput phenotypic assay system to identify compounds that promote differentiation of the progenitors into mature, functional brown adipocytes. We have recently screened collections of human proteins as well as non-human biologicals, and have identified several compounds that strongly recruit brown adipocytes. Here we propose to study the molecular mechanisms of action of the hit compounds. We intend to use microarray transcriptional analysis with bioinformatics and systems biology to identify the pathways involved, followed by enzyme inhibitors and receptor panels to identify specific targets. If the mechanisms of these compounds are believed to be safe, these molecules or similar compounds with improved characteristics could be developed as therapeutics for obesity and/or diabetes. The targets may also serve as the basis for small molecule drug discovery. If the Phase I project is successful, we plan to subsequently advance into animal efficacy studies. Compounds will be synthesized in amounts sufficient for in vivo testing, serum stability will be evaluated, and the compounds will then be studied in appropriate animal models of obesity and type 2 diabetes.